Urokinase-type plasminogen activator receptor is internalized by differentmechanisms in polarized and nonpolarized Madin-Darby canine kidney epithelial cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inos itol-anchored protein urokinase plasminogen activator receptor (uPAR) depen ds on binding of the ligand uPA: plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-densit y lipoprotein receptor family, which are internalized through clathrin-coat ed pits. This interaction is inhibited by receptor-associated protein (RAP) . We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynam in efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin-Darby canine kidney (M DCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate),which selectiv ely up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytos is of uPA:PAI-1 was only deceased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells a nd localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relati ve to polarized MDCK cells was insoluble in Triton X-100 at 0 degrees C, an d by surface labeling with biotin we also show that internalized uPAR was m ainly detergent insoluble, suggesting a correlation between association wit h detergent-resistant membrane microdomains and higher degree of clathrin-i ndependent endocytosis. Furthermore, by cryoimmunogold labeling we show tha t 5-10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupa ncy.